The laboratory mouse is the most widely used mammalian model organism in biomedical research, so a thorough annotation of functional variation in the mouse genome would be of significant value. In this study, we compared sequence variation in a comprehensive list offunctional elements (e.g. promoters, enhancers and CTCF binding sites) across 17 inbred mouse strains. Sequences were derived for ∼300 000 functional elements experimentally identified by the mouse ENCODE project as regulating gene expression in 19 different tissue sources. We aligned sequences for each predicted cis-regulatory element to genomesof 17 mouse strains. This yielded a database comprising ∼5 million aligned sequences, allowing interrogation of sequence variation offunctional elements for each of the 19 tissues/cell types in commonly used mouse strains. We also developed an online tool to visualize the genome around each predicted cis-regulatory element in each tissue context and which allows efficient comparison of variation between any two sets of strains. This will be particularly useful in the context of the Collaborative Cross (CC), which was conceived as a powerful new systems genetics resource to accelerate gene discovery. Comprising a large number of inbred strains derived from eight genetically diverse founders, the CC offers rapid mapping and identification of genes that mediate complex traits. We show that, among the 17 sequenced strains, the setof CC founder strains captures the most variability in the ENCODE elements, further emphasizing the value of this resource.